Mammalian Cell Culture and Transfections
Murine embryonic fibroblasts (MEFs) were derived from E16.5wild-type and
PITPα–/– embryos as previously described(Alb et al., 2003). The mammalian cell lines used in this studywere cultured in DMEM containing 10% fetal bovine serum, 1 U/ml penicillin G, 100 µg/ml streptomycin, and 4.2 µlβ-mercaptoethanol (for 500 ml of complete medium). Cultures were incubated at 37°C and in 5% CO2.
COS-7 cells were transfected using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). Briefly, 24 h before transfection the cells were plated at 50–60% confluency in six-wellplates containing glass coverslips. DNA (1.5–2 µg)was
reconstituted in 100 µl of OptiMEM (Invitrogen), mixedwith 2 µl of Plus reagent, and incubated at room temperaturefor 15 min. In a separate microcentrifuge tube, 3 µl of Lipofectamine was diluted in 100 µl of OptiMEM for eachtransfection. After 15 min, the solutions were mixed and thenincubated for 15 min at 25°C. Cells were washed twice
withOptiMEM and incubated at 37°C with DNA mixture in 1 ml OptiMEMfor 3 h. Subsequently, 4 ml of complete medium was added, andcells were cultured for 18–24 h before processing forimmunocytochemistry. MEFs were transfected using the Amaxa (Cologne, Germany) nucleofector following the manufacturer's directions.
Antibody Reagents
PITP antibodies used in this study included: a PITPβ isoform–specificrabbit polyclonal antibody directed against the C-terminal 25amino acid of PITPβ (generous gift from Bruce Hamilton), a PITPαisoform–specific chicken polyclonal antibody directedagainst the last 15 amino acids of PITPα (Alb et al., 2002),and the NT-PITP- antibody rabbit polyclonal immunoglobulin (Ig)raised against the N-terminus of PITP and that recognizes both PITPα and PITPβ (generous gift of Prof. George Helmkamp, Jr.).
The following primary antibodies were used: a monoclonal antibody directed against actin (Chemicon, Temecula, CA), sheep polyclonal anti-TGN38 Ig (Serotec), monoclonal anti-GM130 antibodies (BDBioscience, San Diego, CA), and murine monoclonal anti-giantinIg (generous gift from Dr. Hans Peter Hauri, Switzerland). Secondaryantibodies used included: Alexa fluorescein isothiocyanate 488(Molecular Probes, Eugene, OR), Cy5-conjugated anti-mouse andfluorescein isothiocyanate– conjugated anti-mouse (JacksonImmunoResearch, West Grove, PA), and goat anti- rabbit, goatanti-mouse, or goat anti-chicken horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch).
Cells were cultured on glass coverslips. Cells were fixed for 15 min with 3.7% formaldehyde in phosphate-buffered saline (PBS),permeabilized in 0.2% Triton X-100 in PBS for 4 min, rinsedonce in PBS, and then preincubated for 30 min in blocking buffer (2% BSA in PBS). Permeabilized cells were subsequently incubatedwith suitable primary antibody appropriately diluted in blockingbuffer for 1 h at room temperature, rinsed four times 5 minwith PBS, and then incubated with the secondary antibodies appropriatelydiluted in blocking buffer for 1 h. Cells were rinsed four timesin PBS, and coverslips were mounted onto glass slides and examinedin a Leica SP2 Laser Scanning Confocal Microscope (Leica, Deerfield,IL). Images were processed with the use of Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA).
In classifying PITP localization profiles as “Golgi”, two major criteria were applied. First, for to score a profileas Golgi the appropriate query profile (GFP or PITP) must exhibitobvious and predominant colocalization with a Golgi marker (TGN38or GM130). Second, the Golgi component of the query profilemust be the strongest signal recorded in the cell being scored.Failure to satisfy both these criteria resulted in a non- Golgi score. Fixed and stained samples were blinded before scoring to control for investigator bias.
Pharmacological Challenge
PITPα–/– MEFs were grown on glass coverslips to subconfluency and intoxicated with chelerethryne chloride (0.66 µM; Sigma, St. Louis, MO) or G109203X (10 nM; Sigma) for appropriate times. PKC activity in MEFs was also stimulated by exposure of cells grown on coverslips to PMA (100 nM, Sigma) for 15 min in serum-free medium. Cells were subsequently fixed for PITPβ immunostaining as described above. Cell-free
extracts were prepared for parallel-treated cultures and processed for immunoblot analysis as described below.
SDS-PAGE and Immunoblotting
Cultures were rinsed with ice-cold PBS and scraped into lysis buffer (20 mM Tris-HCL, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mMNaF, 1% Triton 1 mM orthovanadate supplemented with a cocktailof protease inhibitors (Complete; Roche, Indianapolis, IN).For preparation of cell-free extracts, cells (grown to confluencyin a 100-mm dish) were incubated with 700 µl of lysisbuffer at 4°C for 10 min and then scraped with a rubberpoliceman into microcentrifuge tubes. After centrifugation at 14,000 xg for 10 min, the supernatant was mixed in Laemmlisample buffer and heated
for 5 min at 95°C. Samples wereresolved by SDS-PAGE (10%) and transferred to nitrocellulose(Millipore, Billerica, MA). Membranes were blocked overnightat 4°C in TBST (5% dry nonfat milk in 0.05% Tween 20 inTris-buffered saline) and then
incubated for 3 h at room temperaturewith the appropriate primary antibodies diluted in TBST. Membraneswere rinsed four times for 5 min each with TBST and then incubated with the appropriate HRP-conjugated secondary antibody for 1h, and washed four times for 5 min each with TBST. Blots weredeveloped on x-ray film (Eastman Kodak,
Rochester, NY) using the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham, Arlington Heights, IL).
Generation of PITPα-GFP and PITPβ-GFP cDNAs
PCR primers for rat-PITPα and rat-PITPβ cDNA sequences were flanked on the 5' end with the restriction enzyme site HindIII and onthe 3' end with the restriction enzyme site BamHI. The HindIII-BamHIPCR fragments were cloned into the pEGFP-C1 plasmid
(Clontech,Palo Alto, CA). Yeast plasmids harboring PITPα and PITPβ cDNAs(Skinner
et al., 1993) were used as templates in the PCR reactionsused for generating the
appropriate DNA fragments for cloning.The resulting plasmids were designated pRE772 (PITPβ-GFP) andpRE774 (PITPα-GFP). Primer sequences used are available fromthe authors by request.
Yeast Complementation Assay
Wild-type and mutant PITPβ or PITPβ-GFP cDNAs, as appropriate, were cloned into the multicopy yeast URA3 vector YEplac195 such that the cDNA was expressed either under control of the powerfulconstitutive PGK promoter or the constitutively expressed but weaker SEC14 promoter. This expression vector was transformedinto the sec14-1ts yeast strain (CTY 1-1A, MATaura3-52 his3Δ200,lys2-810 sec14-1ts; (Cleves et al., 1991b) using the lithiumacetate method of Ito et al. (1983). As matched controls, isogenic vectors with either no insert or with SEC14 or PITP
β
cDNA insertswere alsotransformed into the sec14-1ts yeast host strain.Transformants were selected and cultured in uracil-free glucoseminimal medium (Sherman, 1983). Five OD600 equivalentsof each
strain were resuspended in 200 µl Tris-EDTA bufferand serially diluted 10-fold in Tris- EDTA buffer. An aliquot(5 µl) of each dilution was spotted on duplicate YPD agar plates. One plate was incubated at the 30°C (a permissivetemperature for sec14-1ts
mutants) to report unrestrained growthand viability. The companion plate was incubated at 37°C(normally a restrictive temperature for sec14-1ts mutants) toassess phenotypic rescue of sec14-1ts.
Assays were performed using cytosol prepared from the sec14 Δcki1 host strain CTY303 expressing the desired PITP as describedpreviously (Kearns et al., 1998; Phillips et al., 1999; Li et al., 2000; Vincent et al., 2005). Cytosol fractions generatedfrom CTY303 variants expressing Sec14p (positive control) orno PITP (negative control) were
generated and assayed in parallelwith those fractions containing PITPα, PITPβ, or PITPβ
variants.
Site-directed Mutagenesis
The QuickChange kit (Stratagene, La Jolla, CA) was used. Sequences of the various mutagenic primers used are available from the authors by request. All mutant versions generated were verifiedby nucleotide sequence analysis.
Results
Endogenous PITPβ Localizes to the Mammalian Golgi Complex
Previous experiments suggesting a Golgi localization of PITPβ in mammalian cells relied on microinjection of purified fluorophore-modified protein into cells (De Vries et al., 1996) or creation of stable cell lines that overexpress PITPβ (van Tiel et al., 2002). As a result, several key questions regarding PITPβ localization remain. First, it remains to be demonstrated whether endogenous PITPβ is genuinely a Golgi membrane– associated protein. Second, the precise distribution of PITPβ within the Golgi stack also remains to be determined.
Specific localization of endogenous PITPβ was complicated by our observation that antibodies generated against the extreme C-terminal 15- and 25-residue peptides of these proteins, although facile for distinguishing PITPα from PITPβ by immunoblotting, are not satisfactory for immunofluorescence experiments (unpublished data). To
circumvent this issue, we used polyclonal antibodies raised against amino-terminal sequences conserved between PITPα and PITPβ. These antibodies (NT-PITP-antibody) are suitable for immunofluorescence but are not specific reagents in that these recognize both PITPα and PITPβ isoforms in immunoblotting experiments. The specificity issue notwithstanding, we inspected the endogenous PITP immunofluorescence staining profiles obtained with NT-PITP-antibody in an array of cell lines. Swiss 3T3 fibroblasts exhibited a strong perinuclear staining of what appears to be the Golgi apparatus and a diffuse signal in the cytoplasm and the nuclear matrix (Figure 1A). The PITP profiles obtained with Swiss 3T3 cells and NT-PITP-antibody as reporter were typical. Very similar results were also obtained with a variety of other cell lines including astrocytes, primary neurons, and COS-7, HeLa, and HEK293 cells. That the perinuclear PITP staining identifies the Golgi complex is indicated by the coincidence of this profile with that obtained for the cis-Golgi marker GM130 (Figure 1A). As the NT-PITP-antibody immunofluorescence profiles collected with immortalized cell lines represent the sum of endogenous PITPβ and PITPα distribution, and previous studies indicate PITPα localizes to the cytoplasm and nuclear matrix (De Vries et al., 1996), these various localization profiles suggest that endogenous PITPβ targets to Golgi membranes in a variety of cell types.
To visualize endogenous PITPβ in isolation from PITPα, we used NT-PITP- antibody as PITP detector and took advantage of PITPα nullizygous primary cell lines that we had previously generated. The nullizygous MEFs are well suited for these
experiments as these cells are phenotypically indistinguishable from wild-type MEFs and retain unadulterated levels of endogenous PITPβ (Alb et al., 2002; Alb et al., 2003). As
shown in Figure 1B, NT-PITP-antibody decorates an elaborate ribbonlike perinuclear structure in these PITPα–/– MEFs, and this structure is also stained by the cis-Golgi marker GM130. The GM130 and presumptive PITPβ staining profiles are very similar in form, but are not coincident. These data indicate that PITPβ does not localize to cis-Golgi membranes but, rather, localizes to a distinct subcompartment of the Golgi complex (see below). Very little staining of the cytoplasm or nucleus is observed, and staining of the lacelike ER is also evident. These staining profiles were absent when naive preimmune serum was substituted for NT-PITP-antibody in these experiments.
To confirm localization of the known PITPβ, we constructed a PITPβ-GFP chimera, where GFP was fused to the C-terminus of PITPβ. The activity of the PITPβ- GFP chimera was established with a yeast phenotypic rescue assay. This assay capitalizes on previous demonstrations that high-level expression of mammalian PITPs in yeast rescues the growth and secretory defects associated with inactivation of the essential yeast PITP Sec14p (Skinner et al., 1993; Tanaka and Hosaka, 1994). This rescue is dependent on robust PtdIns-binding/transfer by the heterologous mammalian PITP (Alb
et al., 1995). As shown in Figure 1C, a sec14-1ts yeast strain carrying an ectopic copy of the wild-type SEC14 gene grows robustly at 37°C. By contrast, the isogenic sec14-1ts strain fails to grow at all at 37°C, i.e., the restrictive temperature at which the
thermolabile sec14-1ts gene product is inactive. Expression of PITPβ-GFP restored robust growth to the sec14-1ts yeast mutant at the restrictive 37°C temperature.
The functional PITPβ-GFP was expressed in MEFs and the distribution of the chimera was monitored. These localization experiments confirm an unambiguous affinity
of PITPβ-GFP for Golgi membranes in MEFs (Figure 1D) and also in COS-7 cells (see below).
PITPβ Selectively Associates with the TGN
Although both Golgi and ER membranes harbor pools of PITPβ, Golgi
localization predominates and how PITPβ targets to the Golgi membrane system is the focus of this study. To more precisely assign the Golgi subcompartment of residence for endogenous PITPβ, we performed a series of double-label immunofluorescence
experiments. In these experiments, NT-PITP-antibody was used in combination with compatible antibodies raised against markers for specific Golgi compartments. These markers included GM130 for cis-Golgi, giantin for cis- and medial-Golgi, and TGN38 for the TGN. PITPα nullizygous MEFs were used to ensure specific detection of endogenous forms of PITPβ.
As shown in Figures 2, A and B, endogenous PITPβ exhibits little coincidence of staining with the cis-Golgi marker GM130, or the medial-Golgi marker giantin, even though the general profiles for PITPβ and these markers are very similar. Endogenous PITPβ species exhibit a higher degree of colocalization with the trans-Golgi membrane marker TGN38, however (Figure 2C). The predominant localization of PITPβ to TGN membranes is emphasized in a stereo reconstruction of the MEF Golgi apparatus generated from triple-label experiments monitoring PITP, giantin, and TGN38
(Supplemental Video, Figure S1). The rotating image distinguishes giantin staining from the yellow staining that reports colocalization of TGN38 and PITPβ. We infer from these experiments that PITPβ targets predominantly to the trans-aspect of the Golgi stack in MEFs.
During the course of these studies, we noted the existence in the NCBI Protein Database of an uncharacterized PITPβ spliceoform (referred to as PITPβQGQR, as opposed
to canonical spliceoform that we refer to as PITPβ) that is invisible to our PITPβ-specific antibodies in immunoblot experiments (murine form, accession number AAH34676; rat form, AAH61538; human form, AAH31427). This spliceoform is detected by the NT- PITP-antibody, however, and the PITPβ localization profiles described in Figure 2 represent the sum of the PITPβ and PITPβQGQR profiles (these two spliceoforms are both
expressed in MEFs; unpublished data). Defined GFP-chimeras permit localization of each spliceoform in isolation, however. As further described below, we show that both PITPβ-GFP and PITPβQGQR-GFP reporters target efficiently to similar (albeit not
identical) Golgi subcompartments.
PITPβ C-terminal Motifs Necessary for TGN Targeting
The distinctive localization profiles for PITPβ and PITPα are remarkable in light of the high degree of primary sequence identity shared by these PITPs. To map the determinants specifying targeting of PITPβ to the mammalian TGN in an unbiased manner, we constructed a reciprocal series of PITPβ/PITPα hybrid proteins in the context of a functional PITP-GFP chimera. The functional status of key chimeras was confirmed in the heterologous yeast sec14-1ts phenotypic rescue assay (Skinner et al., 1993); described above and in Figure 1C). All chimeras generated were active in the yeast phenotypic rescue assay and were expressed both in PITPα–/– MEFs and in COS-7 cells. The respective intracellular distributions were imaged and quantified for both cell types. In describing the results of the mapping experiments, we present data obtained with MEFs and report the COS-7 data in Supplemental Materials.
The C-terminal 28 PITPβ residues are both necessary for PITPβ targeting to Golgi membranes and are sufficient to efficiently redirect PITPα to that location (Figure 3A). The results were robust because the incidence of Golgi targeting in cells was >90% for PITPβ and the PITPα/β chimera and <5% for PITPα and the PITPβ/α chimera. Representative images for each chimera are shown in Figure 3B. In the imaging
experiments reported herein, we typically identify the Golgi region by surveying the cis- Golgi marker GM130 but confirmed that assignment by costaining with the pan-Golgi marker wheat germ agglutinin and, for key reporter/mutant constructs, by costaining for TGN38 (see below).
Alignment of the PITPβ and PITPα C-terminal primary sequencesidentifies three motifs of greatest divergence between theseisoforms. We refer to these motifs as BOX1, BOX2, and BOX3 (Figure 3C).Mutagenesis experiments, where each individual BOX region fromPITPα was substituted for the corresponding BOX region of PITPβ, demonstrated that PITPβKQE, PITPβQDPK, and PITPβMTD all exhibitedefficiencies of
Golgi localization similar to those recordedfor the PITPβ control (Figure 3, C and D). Thus, no single BOXmotif is essential for PITPβ targeting to Golgi membranes. Wealso observed that swap of any two of the BOX domains from PITPαinto the PITPβ context did not compromise association of PITPβwith TGN membranes (Figure 3, C and D). These data indicatethat the presence of any single PITPβ motif is sufficient for maintenance of PITPβ localization to the Golgi complex. Parallelanalyses of the localization properties of each chimera were also conducted in COS-7 cells with essentially identical results (Supplemental Materials, Figure S2).
To address the dual criteria of necessity and sufficiency, we tested whether any BOX residues sufficient for PITPβ localization to the TGN were capable of redirecting PITPα to the same. To this end, PITPβ BOX1 or BOX3 residues were incorporated into the context of an otherwise wild-type PITPα. The localization profiles of both constructs (PITPαQET and PITPαTSA) fully recapitulated the nuclear and cytoplasmic distribution of the PITPα control (Figure 4A). Thus, neither BOX1 nor BOX3 has an assignable
targeting function on its own in the context of PITPα. However, BOX2 residues, although dispensable for PITPβ targeting to the Golgi complex, increased the efficiency with which an otherwise wild-type PITPα reporter associates with Golgi membranes. That construct (PITPαKGSR) was scored as targeting to Golgi membranes in 51% of the transfected cells analyzed. Although this level of targeting is not as robust as that observed with the PITPβ positive control (>90%), it is substantial when compared with the basal association of the PITPα control with the Golgi complex (ca. 5%; Figure 4A).
When multiple PITPβ BOX motifs were swapped into the PITPα context,an essentially complete redirection of a PITPα reporter to theTGN was observed. Combinatorial incorporation of PITPβ BOX1 andBOX2 residues, BOX1 and BOX3 residues, or BOX2 and BOX3 residues into the PITPα context yielded chimeras that efficiently targeted to Golgi membranes (Figures 4, A and B). For reasons detailed below, we were particularly interested in any role BOX2 or its individual residues may play in the localization of PITPβ to the TGN. In that regard, the dispensability of BOX2 residues for PITPβ Golgi targeting was further emphasized in a swap of BOX2 from PITPα for the PITPβ BOX2 in the context of a PITPα chimera that harbors the C-terminal 28 PITPβ
save 24 of 28 C-terminal residues where the PITPα BOX2 motif is substituted for that of